Retroviruses encode for a protease that is essential for viral replication. The protease cleaves the viral gag and gag-pol polyproteins at a small number of specific sites to release the mature viral structural proteins and enzymes. The substrate selectivity of the proteases is not obvious due to the lack of a clear consensus sequence at the cleavage sites. The function in viral replication and polyprotein cleavage of Rous sarcoma virus (RSV) and Human immunodeficiency virus type-1 (HIV-1) proteases is being investigated by a combination of molecular modeling, mutational analysis, and measurements of catalytic activity. Crystal structures are available of RSV protease, and of HIV-1 protease in the presence and absence of inhibitors. Molecular modeling and analysis of the crystal structures is being used to design mutations in RSV and HIV proteases, and to design and analyse peptide substrates of RSV and HIV proteases. The proteases recognize and bind to at least 7 amino acid residues, designated P4 through P3', where the peptide bond between Pl and Pl' is cleaved. Different types of amino acid are substituted at each position in the peptide substrate and the protease catalytic activity measured in order to understand the molecular basis for substrate specificity. RSV protease residues that are predicted to form the substrate binding site are being mutated to the amino acid type in HIV-1 protease, and the mutants tested for altered specificity. Other RSV protease variants include altering surface loops that differ in HIV protease to test the importance for activity. In each case, the results are analysed in terms of the molecular substrates, and compared with measurements of specificity. Crystal structures will be determined of RSV and HIV protease mutants with altered activities. The structural and biological information will be combined to develop models of other retroviral proteases.